Surebeads protein a g magnetic beads

The chromatin immunoprecipitation assay was performed using a Zymo-Spin ChIP kit (Zymo Research, Irvine, CA, USA, Cat. No. D5210) as previous described^{10 (link)}. Chromatin was crosslinked with 1% formaldehyde for 10 min at room temperature. The crosslinking reaction was stopped at a final concentration of 0.125 M glycine. The prepared nuclei were mechanically sheared using the Bioruptor Pico sonication system (Diagenode, Liege, Belgium) at 4 °C in 30 s on/off for 25 cycles. A small portion of the chromatin solution was reserved as input DNA. The chromatin samples were incubated with the indicated antibodies by rotating for 16 h at 4 °C. SureBeads Protein A/G magnetic beads (Bio-Rad, Cat. No. 161-4013) were incubated at 4 °C for 1 h by rotation to elute the antibody-chromatin complexes. The bead-antibody-chromatin complex was washed three times with chromatin wash buffer (Zymo Research, Cat. No. D5210). The eluted chromatin was decrosslinked with 5 M NaCl, and then, the chromatin proteins were degraded with proteinase K (genDEPOT, Katy, TX, USA, Cat. No. P2170). ChIP DNA was purified using a QIAquick Spin column (Qiagen, Hilden, Germany, Cat. No. 28115). The ChIP DNA and input were analyzed via qPCR using primer pairs specific to the target gene promoters. The primer sequences are listed in Supplementary Table 2.

Briefly, samples were extracted from control and irradiated H9C2 cells (10 cm dish per sample) using NP-40 (Beyotime, China). Co-IP experiments were performed using SureBeads Protein A/G Magnetic Beads (Bio-Rad, CA, USA). Briefly, 30 μL of SureBeads protein A and G was magnetized and washed with 0.1% PBS/Tween 20 (PBST) and then incubated with 2 μg anti-acetyllysine rabbit pAb (Jingjie PTM BioLabs Inc., Hangzhou, China) or IgG rabbit antibody (Abcam, USA) at room temperature for 30 min. Next, the incubated beads were washed five times with 0.1% PBST and then incubated with 100 μg of extracted protein overnight at 4°C. Proteins were electrophoresed on SDS-PAGE gels and immunoblotted on a PVDF membrane (Millipore, USA). Membranes were incubated with Atp5f1c rabbit antibodies (1 : 1000; Proteintech, Wuhan, China) and detected using a peroxidase-conjugated secondary antibody (1 : 20000; Abcam, USA) with ECL Blotting Substrates (Beyotime, China). Membranes were visualized by chemiluminescence (Bio-Rad, USA) and quantified using the ImageJ 14.9 software (ImageJ, Marlyand, USA).

The open reading frame of human SIRT1‐7, TIP60, and GPAT3 (Miaolingbio) were subcloned into pcDNA3.1‐3×flag or pcDNA3.1‐HA vectors. Transfected cells were lysed with NP‐40 lysis buffer (Biosharp, Anhui, China). Anti‐AcGPAT3 were custom‐made from GL (GL Biochem, Shanghai, China) and applied for IP and Co‐IP assays. The immune precipitants eluted from SureBeads™ Protein A&G Magnetic Beads (Bio‐Rad Inc., Hercules, CA, USA) were examined by immunoblotting as described.
³² (link) Anti‐rabbit and mouse IgG (Beyotime) were used as negative controls. Anti‐TIP60, anti‐SIRT3, anti‐GPAT3, anti‐Flag, and anti‐HA (1:1000) were obtained from Abcam (Cambridge, MA, USA).